Culturing primary human cell of both normal and malignant origin is a highly desirable but difficult task. The major reason is that most defined culture media were selected to maintain a well-defined specific cell type. These media are although rich in all necessary low molecular weight compounds they often lack critical growth and survival factors. Formulating cell culture media often required that it should be possible to produce in very large quantities for relatively low price. However recent introduction of new microfluidic and automated microscopy techniques make these requirements obsolete.
Primary cultures of normal cells can serve as a source for stem cells for e.g. reconstitution therapy or as ex vivo target for gene delivery in gene therapeutic interventions. Primary cultures of tumor cells may be used for large scale drug sensitivity assays in order to identify suitable anti-cancer drugs for a given patient, test the efficacy of new drug candidates to previously untested tumor types and even in screening of compound libraries to identify new anti-cancer lead candidates.
An important feature of human tumors is that they are continuously evolving and the ideal therapy would require individualized measures perfectly matching the biological features of the tumor. The most straightforward way to achieve this goal is to create a sophisticated albeit highly robust and reliable methods that can measure the response-rate of the tumor to a large variety of therapeutic interventions. These measurements should provide the theoretical underpinning and practical evidence for assay-guided therapy.
Modern day treatment protocols are the results of carefully controlled clinical trials involving thousands of patients over many years. During the last decades, we have witnessed an impressive improvement in patient survival in several forms of leukemias and lymphomas. The development was the most impressive in the case of pediatric leukemias. For these diseases, the careful grouping of patients according different biological markers and prognostic indicators proved to be crucial for selection of the most effective treatment protocols. These protocols heavily rely on the use of relatively old drugs developed in the late 70s and early 80s. Key factor to achieve the greatly improved survival was the rigorous adherence to the standardized treatment protocol. This strategy, albeit very successful in terms of saving lives of pediatric patients, resulted in very aggressive treatment with significant toxicity. Moreover, this strategy left little room for introduction of new drugs. This is particularly unfortunate considering that there are more than sixty licensed drugs with potential anti-cancer effects and there are over hundred novel drugs in the development pipeline of the pharmaceutical industry.
On the other hand, in the case of adult leukemias, the major breakthrough originated from the introduction of novel drugs to treat near terminal patients (e.g. bcr-abl inhibitor Gleevec against CML; proteasome inhibitor Bortezomib against multiple myelomas or monoclonal antibodies such as anti CD20—Rituximab against a variety of B-cell tumors). Despite all effort, a sizable fraction of both pediatric and adult patients does not respond to the therapy or experience repeated relapses. In these cases, selection of individually designed treatment protocol and application of novel drugs would be highly justified
The idea to use in vitro drug sensitivity assays to determine the sensitivity profile of leukemic cells was already introduced forty years ago. A variety of techniques were developed that aimed to determine the viability of tumor cells after in vitro culturing with relevant drugs [1]. All assays involve the same four basic steps: i) isolation of cells; ii) incubation of cells with drugs in tissue culture medium; iii) assessment of cell survival; iv) interpretation of the results. Some of these techniques are widely used today such as the ATP conversion, MTT reduction (tetrazolium salt conversion to insoluble formazan) or FMCA (fluorometric microculture cytotoxicity) assays [2, 3] and has been proven to provide clinically useful information about the drug sensitivity of the tumor cells.
The current assays though suffer a number of limitations. One of the most serious limitations is the difficulty to keep primary human tumor cells alive in vitro [4]. Most eukaryotic cells require two types of signals simultaneously for efficient ex vivo expansion: growth-promoting (proliferation) and survival (anti-apoptotic) signals. These signals are conferred as soluble ligands, alternatively ligands that are part of the extracellular matrix or are associated with the surface of neighboring cells. The signaling molecules target receptors on the cells that are specific for any given cell type and differentiation stage. Classical cell culture media normally has to be supplemented by serum or tissue extracts in order to support efficient in vitro cell growth. The regularly used 10% fetal bovine serum provide sufficient amount of PDGF and insulin like growth factors to support the expansion of primary fibroblasts and mesenchymal stem cells. Most primary cells, however, require the addition of extra growth factors. The generation of in vitro adapted cell lines is achieved by selection of rare genetic variants that are able to expand solely with the help of PDGF and IGF like molecules. Both normal and tumor cells of the lymphoid/myeloid lineage require extra growth/survival factors for in vitro expansion. Importantly shortage of survival factors sensitizes the cells for cytotoxic drug effects creating an undesirable overestimation of the drug efficacy in short term in vitro survival assays. The present day assays often require a sizable amount of tumor cells and they are often very labor-intensive. Due to the limited number of measurements they can carry out, most assays do not allow testing of large number of drugs in different concentrations in repeated series. Most assays have low information content and regularly restricted to single measurement readout (e.g. estimation of the number of live cells per sample).
Moreover, not all drugs are suited for the conventional in vitro survival assays. Some of them, such as the highly fluorescent anthracyclines, are incompatible with direct fluorescence readout technology. Despite of these shortcomings short-term survival assays already started to deliver clinically important information [1, 5-7].
Until recently, in vitro assays were regarded as an unreliable approach to predict in vivo drug response. These sentiments stemmed from the unimpressive results of in vitro clonogenic assays of ovarium carcinomas. However, the perspective of in vitro assays is shifting in the prediction of drug response, as is evidenced by in a recent review [8] of most of the work, concerning ex vivo drug sensitivity assays, published through the 1980s—“reflected the prevailing view of cancer as a disease of dysregulated cell proliferation. The description of apoptosis and programmed cell death, fundamental to our modern understanding of human tumor biology, did not occur until well after the heyday of in vitro chemosensitivity testing. By incorporating the modern tenets of carcinogenesis associated with perturbations in cell survival we can now re-examine laboratory assays of drug response in the context of drug-induced programmed cell death.”
The main reason for failures was that most cell culture media were developed by starting out from chemically defined simple solutions and gradually adding different serum or tissue extracts until the test cells showed satisfactory survival and proliferation. It is clear now that new approaches are necessary to revive in vitro chemosensitivity testing, to allow finding the optimal, and preferably personalized, treatment for cancer.
The rapid development of digital imaging, the recent breakthroughs in microscopy and laser optics and new methods in lab automation and microfluidics brought this idealized method within reach. We have shown that it is now possible to build and program automated intelligent confocal microscopes that can test the effect of all presently available anti-cancer drugs on primary tumor cells isolated from volumes as small as 1 ml blood, bone marrow, ascites fluid and pleural effusion. The ability to manufacture 384 well drug plates using preprogrammed fluid dispensing robots allows testing of a large number of drugs in different concentrations, alone or in different combinations. Sophisticated fluorescence labeling techniques reveal multiple vital parameters of the drug treated tumor cells such as viability, cell cycle distribution, metabolic activity and motility.
The only missing element for comprehensive in vitro drug testing is a reliable, efficient culturing medium that preserves the original in vivo conditions as much as possible to help identify the best possible treatment regimen.
To satisfy this need, we have completely re-designed the culture conditions for primary tumor cells. In our approach, we preserve the original in vivo conditions as much as possible. The theoretical basis of our new cell culture medium relies on the following previous observations: primary human lymphocytes, highly apoptosis prone when explanted into cell culture media in the absence of extra growth factors, can survive in total blood for several days [9-12] even if it undergoes partial hemolysis [13]. However, none of these prior art discloses or even suggests the feasibility whole blood as the basis of culture medium for culturing human cells.
Accordingly, the present invention provides a cell culture medium for culturing human cells comprising an anti-coagulated total blood material wherein the hemoglobin level is from about 8 to about 16 g/dl.
In another embodiment, the invention provides a cell culture medium, wherein the cells present in the blood are disrupted and the insoluble remnants of the lysated cells are removed.
In a further embodiment, the invention provides a cell culture medium, wherein the blood is of mammalian origin. In a specific embodiment, the invention provides a cell culture medium, wherein the blood is of human origin.
In another embodiment, the invention provides a cell culture medium, wherein the ion content of the medium is restored to match that of the extracellular fraction of total blood starting material.
In a further embodiment, the invention provides a cell culture medium, wherein the low molecular weight (<3000 Dalton) nutrient content and/or composition is essentially the same as that of a conventional cell culture medium selected preferably form the group consisting of RPMI, DMEM, and IMDM.
In another embodiment, the invention provides a cell culture medium, wherein the reducing capacity of the medium is restored by addition of reduced glutathione.
In a further aspect, the invention provides the use of a cell culture medium according to the invention for culturing human cells.
In a further aspect, the invention provides a method for the preparation of a cell culture medium for culturing human cells, comprising                (a) providing anti-coagulated mammalian blood as starting material;        (b) mechanically disrupting the cells present in the blood;        (c) removing the insoluble remnants of the lysated cells.        
In another embodiment, the invention provides a method, wherein the mechanical disruption of the cells is achieved by freezing the total blood or by ultrasound, electric blade homogenizer or French press.
In another embodiment, the invention provides a method, wherein removal of the insoluble remnants of the lysated cells is achieved by ultracentrifugation or mechanical filtration.
In another embodiment, the invention provides a method, wherein the low molecular weight (<3000 Dalton) content and/or composition of the medium is adjusted by dialysis against conventional cell culture medium selected preferably form the group consisting of RPMI, DMEM, and IMDM.
In another embodiment, the invention provides a method, wherein the reducing capacity of the medium is restored by addition of reduced glutathione.
In a further aspect, the invention provides a cell culture medium for culturing human cells, obtainable by the method according to the invention.